1. Field of the Invention
The present invention relate to the field of biotechnology and molecular biology research. The present invention relates to peptides highly efficient in inhibiting telomerase activity and methods of preparation and uses thereof.
2. Background Art
Telomerase is a ribonucleoprotein for synthesizing and extending chromosome telomeres in cells. It contains two basic components: a reverse transcriptase catalytic subunit hTERT and an RNA component hTR. Telomerase can use its own RNA as a template to reverse transcribe and synthesize telomere repeat sequences, which are added to the ends of chromosomes to compensate for the loss of telomeric DNA during cell divisions and maintain telomere lengths, Studies show that telomerase activity is almost undetectable in normal human cells. Therefore, the number of normal somatic cell division is limited. Telomeres are shortened 50-200 bp after every cell division. When telomeres are shortened to a certain degree, cell growth is inhibited, known as cellular senescence, leading to cell death. However, telomerase activities can be detected in the majority of malignant tumor cells (85%) and these activities are relatively high. The re-synthesis of telomere compensates for the continuous telomere loss during cell division processes and enables cells to divide continuously. This is an important mechanism for cell immortalization and tumorigenesis.
Kim et al., analyzed and summarized a large amount of research results, by examining more than 100 malignant tumor samples, and showed that the sensitivity of using telomerase in the diagnosis of tumor was 85%, specificity was 91%, the positive predictive value was 91%, and the negative predictive value was 81%. This fully demonstrates the value of using telomerase in diagnosis of tumors. (Kim N W, Piatyszek M A, Prowse K R, et al. Specific Association of human telomerase activity with immortal cells and cancer. Science. 1994 Dec. 23; 266 (5193): 2011-5.). Telomerase activation is considered a major factor for malignant tumor formation. The levels of activation and expression are closely associated with tumorigenesis and metastasis. Telomere shortening by inhibiting telomerase is considered a mechanism for cancer cell suppression. Thus, telomerase becomes an ideal target for targeted tumor therapy. Telomerase inhibitors for the treatment of tumors are being developed by many companies. Among these, GRN163L has begun a phase 2 clinical trial, and several telomerase vaccines will soon complete their clinical trials and enter the market.
LPTS (Liver Putative Tumor Suppressor) is a liver-related new candidate tumor suppressor gene obtained from normal human liver cDNA library using positional cloning approach by the inventors [C. Liao, M. J. Zhao, H. Song, K. Uchida, K. K. Yokoyama, T. P. Li, Identification of the gene for a novel liver-related putative tumor suppressor at a high-frequency loss of heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma. Hepatology 2000, 32 721-727]. LPTS gene is located on human chromosome 8, region 8p23, which is lost at a high frequency in a variety of malignant tumor cells. Studies show that the expression levels of LPTS in hepatocellular carcinoma tissues and hepatoma cell lines are extremely low or undetectable. Introduction of LPTS gene into liver cancer cells can inhibit their growth, proliferation, and eventually induce liver cancer cell death. [Liao C, Zhao M J; Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma. World J Gastroenterol, 2003, 9:89-93]. LPTS gene has been granted a patent in China in 2004 (Zhao Mujun et al.: “a liver cancer-related gene and use thereof,” Patent No.: ZL 00115395.1, Patentee: Shanghai Institute of Biochemistry, Chinese Academy of Sciences; issued date: Oct. 13, 2004). In 2001, Dr. Lu's laboratory reported another full-length LPTS gene transcript PinX1 and found that PinX1 encoded protein can bind to the catalytic subunit of telomerase hTERT and inhibit telomerase activity [Zhou X. Z., Lu K. P; The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor. Cell, 2001, 107, 347-359]. Based on this mechanism, it was proven, for the first time, that LPTS/PinX1 is a natural telomerase inhibitory protein that can inhibit tumor cell proliferation, providing a new way for targeted tumor therapy. In 2005, the present inventors submitted a patent application related to LPTS protein preparation (Zhao Mujun et al.: “Preparation and purification of telomerase activity inhibitory protein,” Patent Application No.: 200510030526.5, filing date: Oct. 14, 2005; Applicant: Shanghai Institute for Biological Sciences, Chinese Academy of Sciences). That patent application provides a method for preparing the LPTS protein (LPGENE1) and an active telomerase inhibitory LPTS fragment LPTS133-328 (LPGENE2), and shows that the active LPTS telomerase inhibitory fragment is located at the C-terminal amino acid residues 133-328. A patent application submitted in 2008 by the present inventors showed that TAT and LPTS133-328 fusion protein (Patent Application Number: 200810041324.4) can pass through cell membranes and has an excellent efficacy in the inhibition of tumor cell growth.
Given that LPTS protein is an important protein closely associated with tumor cell growth, it is therefore necessary to further study LPTS and to develop more effective tumor inhibitory drugs to meet the need of clinical applications.